Project Summary/Abstract Oceanic harmful algal blooms (HABs) pose a major environmental threat to human health, coastal ecosystems, and marine food supplies. Due to climate change, HABs are anticipated to continue increasing in severity and frequency, impacting millions in coastal communities. The largest HAB ever recorded, comprised primarily of toxic Pseudo-nitzschia diatoms, spanned the North American west coast from Alaska to the Baja peninsula during 2015. High levels of the neurotoxin domoic acid (DA) produced by this bloom resulted in major fishery closures in the Pacific Northwest to protect human health. Although bloom monitoring and toxin detection is quite sophisticated, relatively little is known about the underlying basis for toxin production among marine HAB species. Understanding the regulation and expression of the genes encoding HAB toxin biosynthesis could improve bloom monitoring by introducing a genetic component to help predict a bloom?s potential for toxicity. The goal of this proposal is to characterize the cellular and transcriptional regulation of DA biosynthesis by using laboratory model systems, cultured Pseudo-nitzschia spp. isolates, and environmental samples. My recent work has uncovered the clustered genes that encode the DA biosynthetic pathway in Pseudo-nitzschia. I now plan to extend my research to study DA biosynthesis (dab) genes in diatom systems. By describing where DA biosynthesis takes place in the cell, we can place DA biosynthesis in the context of overall cellular physiology. In addition, tracking dab gene expression both in culture and in environmental samples can help us correlate gene transcription to bloom toxicity. I hypothesize that a full characterization of the cellular and transcriptional regulation of toxin biosynthesis will enable us to monitor Pseudo-nitzschia HAB toxicity at the transcript level. Our findings will also help the community understand the oceanic conditions that induce DA production in blooms. To achieve these goals, I will explore the subcellular localization of the Dab enzymes using the model diatom Phaeodactylum tricornutum as a heterologous host using diatom-specialized molecular techniques. Next, I will explore dab gene expression in isolates of Pseudo-nitzschia spp. grown under a variety of DA-inducing culture conditions to explore environmental inputs to toxicity. Finally, I will apply our findings from the culturing experiments to the larger environment by generating metatranscriptomics datasets from weekly filter samples collected in Monterrey Bay before, during, and after the 2015 North American Pseudo-nitzschia bloom. This proposal is designed to supplement my prior research on DA biosynthesis, allowing me to pursue further academic training opportunities together with my Ph.D. co-advisors, Professors Moore and Allen. My advisors have been chosen for their diverse and complementary scientific expertise in order to cover all elements of the proposed research.